The goal of this research is to define molecular events that underlie bacterial chemotaxis. These events allow the bacteria to generate a memory of their past environments and compare that memory to their current environment. The bacteria then modulate the sense of rotation of their flagellar motors to extend runs in favorable directions (CCW-counterclockwise) or turn (CW-clockwise rotation) into a new, essentially random direction. Chemotaxis is characterized by extremely high sensitivities to small temporal changes in attractant or repellent concentrations. This high gain (several hundred fold) is generated by two highly cooperative molecular machines acting in series; the flagellar switch and signaling complex of chemotaxis receptors, CheA kinase and the coupling protein CheW. These two sources of gain are the focus of this proposal. The cell's adaptation system extends this high gain response to over a factor of ~105 in attractant and repellent concentration. Adaptation is primarily associated with the covalent modification of the chemotaxis receptors by methylation of specific glutamate residues although it has recently been shown that changes in the number of certain motor components can contribute to long-term adaptation. Two central questions in bacterial chemotaxis are still poorly understood; (1) How does the receptor and CheW regulate CheA activity and what structural changes are associated with receptor adaptation? (2) What is the structure of the flagellar switch and what changes are associated with the switch from CCW to CW rotation. Experiments designed to investigate these two largely unsolved questions are the foci of this proposal. These experiments include in vitro measures of information propagation from the ligand binding sites in the chemotaxis receptors using modern NMR and EPR experiments. Similar techniques will be used to probe structure and conformational changes associated with the membrane associated signaling complex of receptors, a coupling protein, CheW, and the chemotaxis kinase, CheA. We will use water dynamic nuclear polarization by nitroxide spin labels to probe solvent exposure and dynamics in different signaling states of the complex. We will use a combination of mutational analysis and chemical cross linking to establish the domain organization of the flagella switch component FliG in assemble flagella and its precursors. Diffraction quality crystals of the complex of FliG and the C-terminal domain of the membrane protein FliF have been obtained and we prose to complete the structure of that complex to understand the first step in flagellar assembly.